The present invention relates to a DNA fragment coding for a modified nuclear glucocorticoid receptor (GR), and a DNA fragment coding for a ligand binding domain (LBD) of said receptor, as well as a DNA fragment coding for a fusion protein comprising said receptor of said ligand binding domain of said receptor fused to a protein whose activity is induced in the presence of glucocorticoid ligands.
The present invention also relates to a modified nuclear glucocorticoid receptor, in particular the human receptor and its modified ligand binding domain, as well as a fusion protein comprising said receptor or said ligand binding domain.
The present invention also relates to a vector for conditionally expressing a protein, in particular a foreign protein, in human or animal host cells, in particular mammalian cells. The present invention also relates to a method of expressing a protein in said cells. Moreover, the present invention relates to a method for the conditional excision, insertion, inversion or translocation of a DNA fragment in human or animal, in particular mammalian, host cells.
The present invention relates in addition, to a vector for transferring a DNA fragment into said human or animal, in particular mammalian, host cells and to its use as a medicament, as a tool for analyzing and studying the function of a gene as well as to a method of treating cells ex vivo or in vitro.
Finally, the present invention relates to human or animal cells transfected with an expression and/or transfer vector according to the invention as well as to transgenic animals derived therefrom.
The glucocorticoid receptor (GR) is a protein for regulating transcription of genes which mediate the action of glucocorticoids in target cells. The GR and other members of the super family of nuclear receptors (ligand-activated transcription factors) exhibit a common modular structure. The variable N-terminal region contains the constitutive transactivation activity AF-1. The core DNA binding domain (DBD) is highly conserved between various species and allows the binding of the receptor to its related DNA response element (GRE in the case of GR). The ligand binding domain (LBD), located in the C-terminal region of GR, not only binds the ligand, but also comprises multiple distinct activities: a surface for interaction with hsp90 and a distinct surface for homodimerization, a nuclear localization signal and a ligand-dependent transactivation function AF-2 (for review articles and references, see (1-5)). The LBD domain is highly conserved between various species. A transactivation function AF-2 has been identified in the C-terminal part of various LBDs (6-10). The integrity of the core part of the region containing AF-2 is required for the ligand-dependent transcription activation by the corresponding nuclear receptors, and for the interaction between this region and related transcriptional intermediate factors (TIF, also called coactivators or mediators) (11-19). A general model has been proposed (19) in which the binding of the ligand induces a conformational change in the ligand binding domain by calling into play in particular the C-terminal region harboring the core part of AF-2, thus generating a surface for an effective interaction with the TIFs. The LBDs of the nuclear receptors contain conserved regions possessing a canonic structure In the form of a helix. These structural data have made it possible to carry out a common alignment or the LBDs of all nuclear receptors. The H12 helix contains the transactivation function AF-2 (19). The m no acids of helices H11 and H12, in particular of the glucocorticoid receptors from various species such as humans, rats, mice and xenopus, are conserved.
Several mutations in the LBD of GR have been previously described. Two major types of mutation can be distinguished:
the first type consists in mutations which positively or negatively affect the affinity for binding to the ligand. For example, the replacements of the cysteine residue 656 by glycine in rat GR (20) and of the methionine residue 565 by arginine or of the alanine residue 573 by glutamine in the human GR (hGR) (21) increase the affinity for binding to the ligand and lead to a shift in the dose-response curve for the transactivation in the direction of the lowest ligand concentrations. These mutants are consequently designated xe2x80x9csuper GRxe2x80x9d. Likewise, mutants have been reported which have a lower affinity for binding to the ligand (21-24). LBD mutations which shift the ligand dose-response curve in the direction of the highest ligand concentrations result from an altered hormone binding affinity (25-26);
the second group of LBD mutants comprises those which affect the transcriptional activation function (AF-2) without altering the binding to the ligand. As examples, there may be mentioned mutations in the region containing AF-2 which are linked to a loss or a decrease in the transactivation potential, but do not affect the affinity for binding to the ligand (6-10).
The fusion of the ligand binding domain (LBD) of nuclear receptors to heterologous proteins has made it possible to control their activity in numerous cases.
The activities of Myc, c-Abl, Src, erbB1, Raf, E1A Cre and FLP may be modulated by producing fusion proteins with the LBD of nuclear receptors (27-29). However, the ligands for these receptors are present in numerous biological systems and are thus capable of inducing a basal activity level. To avoid such problems, a mutated LBD of the estrogen receptor (ER) has been fused to the c-Myc protein (30), or to the Cre and FLP recombinases (28 and 29).
The recombinases of the family of xcex integrases catalyze the excision, insertion, inversion or translocation of DNA fragments at the level of specific sites of recognition of said recombinases (31-36). The recombinases are active in animal cells (35).
The Cre recombinase, an integrase of 38 KDa from bacteriophage P1, catalyzes recombination between two DNA sequences of 34 base pairs called loxP in the absence of cofactors (32, FIG. 1).
The position on one or more DNA molecules and the orientation of loxP sites relative to each other determine the type of function of the recombinase, excision, insertion, inversion or translocation, in particular the Cre recombinase (FIG. 1). Thus, the recombinase activity of Cre is an inversion when two LoxP sites are head-to-tail on the same DNA fragment and an excision when the LoxP sites are a direct repeat on the same DNA fragment. The recombinase activity is an insertion when a loxP site is present on a DNA fragment, it being possible for a DNA molecule such as a plasmid containing a loxP site to be inserted at the level of said loxP site (FIG. 1). The Cre recombinase can also induce a translocation between two chromosomes provided that a loxP site is present on each of them (37) (FIG. 1). More generally, the Cre recombinase is therefore capable of inducing recombination between one or more different DNA molecules provided that they carry loxP sites.
Likewise, the FLP recombinase, a recombinase of 43 KDa from Saccharomyces cerevisiae, is capable of the same type of action on DNA fragments containing recognition sites FRT (34).
By producing a fusion protein (chimera) between the Cre recombinase and the C-terminal region of the human receptor for estrogens containing a Valine at position 400, a molecule is obtained which is capable of excising, in the presence of the receptor ligand, estradiol, the DNA sequences located between two loxP sites, as well as one of the loxP sites, when the latter are a direct repeat, whereas in the absence of ligand, the excision does not take place (28).
The activity of the FLP recombinase can also be regulated by producing chimeras between the FLP and the binding domain of nuclear receptors. By fusing the FLP with the LBD of the estrogen receptor or of the glucocorticoid receptor in the absence of ligand, the recombinase activity is very low, whereas it is rapidly induced by the respective ligands for the LBDs (29).
However, the use of fusion proteins between recombinases and LBDs of nuclear receptors to excise, in a controlled manner, DNA fragments situated between sites of recognition of said recombinases can pose a problem, given that the ligand concentrations present in animals or in cell culture media may be sufficient to induce, at least partially, the recombinase activity.
The present invention provides a recombination system inducible in animals, in particular mammals, by virtue of the use of a fusion protein produced between a recombinase and the ligand binding domain derived from the glucocorticoid receptor whose activity is inducible by synthetic glucocorticoids but not by natural glucocorticoids in particular at concentrations of less than 10xe2x88x926 M, that is to say physiological concentrations.
To avoid induction of the recombinase activity of the chimeric protein by the endogenous ligands, the present invention indeed proposes to provide a mutation in the LBD of the glucocorticoid receptor allowing It to be more strongly activated in the presence of synthetic glucocorticoid ligands than in the presence of natural glucocorticoid ligands at physlologlcal concentrations.
The subject of the present invention is indeed a modified nuclear glucocorticoid receptor, in particular mutated in the region of the ligand binding domain, so that the activity of said receptor is more strongly inducible by a synthetic glucocorticoid ligand than by a natural glucocorticoid ligand.
According to the present invention, xe2x80x9c(mutated) glucocorticoid receptor more strongly inducible by a synthetic glucocorticoid ligand than by a natural glucocorticoid ligandxe2x80x9d is understood to mean that the activity of the mutated receptor according to the invention is induced more strongly by synthetic glucocorticoid ligands than by natural glucocorticoid ligands at a given concentration and that the mutated glucocorticoid receptor according to the invention is a lot less sensitive to the natural glucocorticoids than the wild-type receptor.
xe2x80x9cActivity of said receptorxe2x80x9d is understood to mean here the transcriptional activity of said receptor and/or, where appropriate, the activity of regulating the activity of a protein fused to said receptor or to said ligand binding domain, induced in the presence of glucocorticoids. There may be mentioned more particularly the activity of a recombinase protein fused to said receptor or to said ligand binding domain of said receptor.
xe2x80x9cNatural glucocorticoidsxe2x80x9d are understood to mean here steroid hormones secreted by the cortex of the adrenal glands such as cortisol, corticosterone or aldosterone. xe2x80x9cSynthetic glucocorticoidsxe2x80x9d are understood to mean chemically synthesized agonists of said receptor for the relevant activity. There may be mentioned, among the letter, dexamethasone, triamcimolone acetonide RU 486, RU 28362, bimedrazol, fluocinolone acetonide.
Some synthetic ligands will be agonists for inducing said particular activity of the receptor and antagonists in relation to another activity of the receptor. Thus, the compound RU 486 induces the activity of a recombinase protein fused to said receptor or to said ligand binding domain of the receptor but does not induce the transcriptional activity of the receptor.
The synthetic glucocorticoid ligand may bind to the (nonmutated) wild-type nuclear receptor, but it is possible that said synthetic ligand binds to the mutated receptor and not to the natural receptor. It is also possible that if said synthetic ligand binds to the wild-type receptor, the activity of the latter is nevertheless not induced by binding to the synthetic ligand.
A mutation has in particular been identified in the LBD domain of the nuclear glucocorticoid receptors (isoleucinexe2x86x92threonine) situated between helices H11 and H12 (19). This mutation causes a loss of activity for the receptor in the presence of natural glucocorticoids. More precisely, this glucocorticoid receptor mutated between H11 and H12 has no or very little transcriptional activity in the presence of natural glucocorticoids at their natural physiological concentrations, in particular concentrations of less than 10xe2x88x926 M. Its activity may, however, be induced by synthetic glucocorticoid ligands, in particular dexamethasone (dex).
The subject of the present invention is more particularly a mutated human receptor called hGR (I747/T) characterized in that it has, as sequence, the amino acid sequence of the human wild-type nuclear glucocorticoid receptor with an isoleucinexe2x86x92threonine mutation at position 747, and still more particularly a modified human nuclear receptor which has, as amino acid sequence, a sequence substantially as represented in the SEQ ID NO: 1.
The isoleucine 747 to threonine mutation in the C-terminal part of the ligand binding domain (LBD) of the glucocorticoid receptor alters the capacity of the natural ligands to induce the transactivation activity thereof. Natural glucocorticoids such as cortisol, aldosterone or corticosterone are very weakly active or completely inactive with the GR mutant (I747/T), whereas synthetic glucocorticoids such as dexamethasone (Dex) efficiently stimulate the GR-mediated transactivation (I747/T). However, the corresponding ligand dose-response curve, for the transactivation induced by Dex, is shifted toward the highest concentrations, compared with that obtained with the wild-type (WT) GR. Neither the shift nor the inability of cortisol to efficiently activate GR (I747/T) are due to an altered affinity for binding to the ligand in vitro.
Indeed, the mutation in the LBD of GR (I747/T) does not substantially alter the affinity for binding to the ligand in vitro. However, this mutant exhibits a substantial shift compared with the WT GR for the activity curve toward the high ligand concentrations.
Moreover, so-called physiological concentrations of natural glucocorticoid ligands which are sufficient to activate the WT GR cause only a residual or zero activity in the mutant GR.
This mutant differs from the other GR LBD mutants previously described (22, 38) whose altered activation function is closely correlated with a modification in their properties for binding to the ligand in vitro.
The subject of the present invention is also a ligand binding domain of the nuclear glucocorticoid receptor according to the invention and particularly the LBD domain [GR(I747/T)], characterized in that it has an isoleucinexe2x86x92threonine mutation between helices H11 and H12. More particularly, it has, as sequence, the ainmo acid sequence of the ligand binding domain of the human glucocorticoid receptor substantially as represented in SID No. 1 from amino acid 532 to amino acid 777 with an isoleucinexe2x86x92threonine mutation at position 747.
The present invention also provides a DNA fragment encoding a modified nuclear glucocorticoid receptor, in particular mutated in the region of the ligand binding domain (LBD) according to the invention, as well as a DNA fragment encoding a ligand binding domain (LBD) of the modified receptor according to the invention.
The subject of the present invention is therefore more precisely a DNA fragment encoding a wild-type nuclear glucocorticoid receptor with an isoleucine to threonine mutation situated between helices H11 and H12 of the amino acid sequence of said receptor, and still more particularly a DNA fragment, characterized in that it has, as sequence, a coding sequence of the human nuclear glucocorticoid receptor whose amino acid sequence is substantially that represented in SEQ ID NO: 1.
In one embodiment, the subject of the present invention is a DNA fragment, characterized in that it has, as sequence, the cDNA sequence of the human nuclear glucocorticoid receptor substantially as represented in SEQ ID NO: 1 comprising the ACC codon coding for threonine at position 747 of the amino acid sequence of said receptor.
Likewise, the subject of the present invention is a DNA fragment encoding the ligand binding domain (LBD) of the modified nuclear glucocorticoid receptor with an isoleucinexe2x86x92threonine mutation situated between helices H11 and H12 and in particular at a position corresponding to position 747 of the amino acid sequence of the human nuclear glucocorticoid receptor.
More particularly, the present invention provides a DNA fragment encoding the ligand binding domain of the modified human nuclear glucocorticoid receptor, characterized in that it has, as sequence, a sequence coding for the amino acids of the ligand binding domain of the human nuclear glucocorticoid receptor whose amino acid sequence is substantially as represented in SEQ ID NO: 1 from amino acid 532 to amino acid 777.
Still more particularly, the present invention provides a DNA fragment, characterized in that it has, as sequence, the cDNA sequence encoding the ligand binding domain (LBD) of the human nuclear glucocorticoid receptor substantially as represented in SEQ ID NO: 1 from codon 532 to codon 777 with the ACC codon at position 747.
The subject of the present invention is, in addition, a vector system for conditionally expressing a protein, in particular a foreign protein, in host cells comprising an element for control of transcription inducible by a complex formed by a nuclear glucocorticoid receptor and a ligand, characterized in that it comprises:
a first DNA fragment consisting of a DNA fragment coding for said protein under the control of elements ensuring its expression in said host cells, said elements ensuring its expression comprising a sequence for control of transcription (RE) inducible by the receptor according to the invention complexed with a synthetic glucocorticoid ligand, and
a second DNA fragment consisting of a functional DNA fragment coding for said receptor according to the invention or only part of said fragment comprising the region which recognizes the ligand (LBD) according to the invention, and the DNA binding region (DBD) which attaches to said sequence for control of transcription (RE),
it being possible for said first and second DNA fragments to be carried by the same vector or two vectors separately.
Some cells have endogenous GR which may be activated by natural ligands and synthetic ligands. If it is desired to activate a given gene at will in such cells, without it being possible for it to be activated by endogenous ligands or by endogenous receptors, it is necessary for this gene to contain an RE different from GRE and to use a chimeric activator comprising the corresponding DBD and a mutated LBD according to the invention. This gene can therefore be activated by synthetic ligands, but not by natural ligands.
Accordingly, in one embodiment, the DNA binding domain (DBD) of the glucocorticoid receptor is replaced by that of another transactivator, in particular that of the yeast protein Gal 4 and the sequence for control of transcription (RE) of the glucocorticoid receptor is replaced by that of another transactivator, in particular the 17 mer (17m) sequence recognized by the Gal4 protein.
The subject of the present invention is therefore also a method of expressing a foreign protein in human or animal, in particular mammalian, cells, characterized in that cells are cultured which contain an expression vector system according to the invention, and in that said synthetic ligand is added to the culture medium and then the protein synthesized is recovered.
The synthetic glucocorticoid ligands diffuse freely in the cells when they added to the culture medium or injected into an animal.
In this method, a synthetic ligand chosen from dexamethasone, triamcinolone acetonide, RU2836, bimetrazole, deacylcortivazol and fluocinolone acetonide will be used in particular.
The subject of the present invention is, in addition, a fusion protein comprising a receptor according to the invention or a ligand binding domain according to the invention and a protein whose activity is more strongly inducible by binding of said receptor or of said ligand binding domain (LBD) of said receptor with a said synthetic glucocorticoid ligand than with a natural glucocorticoid ligand.
In particular, the subject of the present invention is a fusion protein which comprises a recombinase protein and more particularly of the family of xcex integrases and still more particularly the Cre protein of the bacteriophage P1.
In one embodiment, the fusion protein comprises the C-terminal part of the hinge region D of the human nuclear glucocorticoid receptor, intercalated between the Cre protein and said ligand binding domain of the modified receptor according to the invention.
The actual recombinase activity of the fusion protein is similar to that of Cre. It therefore makes it possible, like Cre, to excise or reverse the DNA sequences located between the loxP sites, to insert a plasmid containing a loxP site at the level of a loxP site located in the genome, or to carry out a translocation between two chromosomes each containing a loxP site.
The subject of the present invention is also a fusion gene coding for the fusion protein according to the invention comprising a DNA fragment coding for a modified nuclear glucocorticoid receptor or a ligand binding domain of said receptor according to the invention, as well as a DNA fragment coding for a protein of which it is desired to regulate the activity, said activity being inducible by binding of said receptor or of said ligand binding domain with a said synthetic glucocorticoid ligand, but not by binding with a natural glucocorticoid ligand when said protein is fused to said receptor or to said ligand binding domain of said receptor.
More particularly, the subject of the present invention is a fusion gene comprising, in the 5xe2x80x2xe2x86x923xe2x80x2 direction:
a DNA fragment coding for the Cre recobinase of bacteriophage P1:
a DNA fragment coding for all or part of the hinge region D of the nuclear glucocorticoid receptor, a region situated between the DBD domain and the LBD domain, and
the DNA fragment coding for the modified, in particular mutated, ligand binding domain (LBD) of the nuclear glucocorticoid receptor according to the invention.
And still more particularly, the subject of the present invention is a fusion gene, characterized in that it has, as sequence, a sequence coding for the amino acid sequence of SEQ ID NO: 2 comprising:
amino acids 1 to 343 which correspond to the Cre recombination;
amino acids 346 to 377 which correspond to the C-terminal region of the D region of the human glucocorticoid receptor;
amino acids 378 to 623 which correspond to the LBD [GR(I747/T)].
In a specific embodiment, the fusion gene has substantially, as sequence, the Cre-LBD[GR(I747/T)] sequence as represented in SEQ ID NO: 2 in which amino acids 626 to 667 correspond to the F region of the human estrogen receptor.
The subject of the present invention is also a vector for expressing the fusion protein encoded by the fusion gene according to the invention, in animal, in particular mammalian, host cells, characterized in that it comprises the fusion gene according to the invention, placed under the control of elements for expression ensuring its expression in said host cells. The vector for expressing the fusion protein may also be a vector or integrating into the genome of the host cells.
In the vectors according to the invention, to express the receptor, the LBD or the fusion protein according to the invention, it is necessary for the DNA fragments coding for these molecules to be placed under the control of a regulatory sequence, in particular of the promoter/enhancer sequences. Promoter/enhancer sequences of the SV40 virus may be used in particular.
The subject of the present invention is, in addition, a method of recombination, in particular of conditional excision, insertion, inversion or translocation at the level of a DNA fragment containing one or two sites for specific recognition of a recombinase protein in human or animal, in particular mammalian, host cells, characterized in that it comprises the steps in which:
1) a fusion protein according to the invention or a vector for expressing said fusion protein according to the invention is introduced into said host cells under conditions allowing the expression of the fusion protein according to the invention and,
2) said fusion protein is complexed with a said synthetic glucocorticoid ligand by exposing said ligand to said host cells.
The present invention therefore provides a method for the conditional deletion of a DNA fragment in which a method of excision according to the invention is used, and in which said DNA fragment to be excised is integrated between two recombinase protein recognition sites oriented as a direct repeat. In particular, said DNA fragment may be chosen so that the excision of said DNA fragment has, as effect, the inactivation of said gene. The excision of said DNA fragment can also allow the synthesis of a functional protein if said fragment contains, for example, a stop codon or a polyadenylation signal.
In the preferred embodiment according to the invention, said recombinase protein specific recognition sites are the loxP sites and said recombinase protein is the Cre protein of bacteriophage P1.
Said DNA fragment which it is desired to recombine, in particular to excise and said recognition site(s) specific for a recombinase protein may be carried by a plasmid or viral vector, or may be integrated into the chromosome of the host cells.
The integration of a gene carrying the loxP sites in a genome may be random or targeted, In particular, the integration of the recombinase protein specific recognition sites, in particular of the loxP site(s) for the Cre recombinase, may take place by homologous recombination of the gene comprising said DNA fragment to be excised or inverted (2 loxP sites) or respectively inserted or translocated (1 loxP site) with a said modified gene comprising said DNA fragment to be excised flanked in 5xe2x80x2 and/or 3xe2x80x2 by said recombinase recognition site(s) depending on the desired application, in particular the loxP sites.
A chimeric protein, Cre-LBD[GR(747/T)] of SID No. 2, composed of the Cre recombinase fused to the LBD of GR (I747/T) was constructed. The fusion protein Cre-LBD[GR(I747/T)] makes it possible to carry out a recombination between the loxP sites, in a mammalian cell, following a treatment with synthetic glucocorticoids. In the absence of treatment, or in the presence of concentrations of natural glucocorticoids of up to 10xe2x88x926 M, no excision is observed. This chimera is capable of excising the DNA sequences situated between the loxP sites previously integrated into the genome of F9 cells (mouse embryo carcinoma) or present on a plasmid after transfection of these cells with a vector expressing Cre-LBD[GR(I747/T)] following a treatment with Dex at 10xe2x88x926 M or 10xe2x88x927 M, whereas at such concentrations, cortisol does not induce recombinase activity. As already stated, the Cre-LBD[GR(I747/T)] expression vector may also be a vector for integration into the aenome of the host cells.
This system therefore makes it possible to release the recombinase activity of a chimeric protein at given and chosen time. The fusion protein Cre-LTD[GR(I747/T)] may be expressed in cells containing loxP sites without modifying the locus containing the loxP sites. Recombination at the level of the loxP sites takes place only after treatment with synthetic glucocorticoids. Furthermore, by expressing the fusion protein Cre-LBD[GR(I747/T)] in an animal under the control of a promoter exhibiting cellular specificity, it is possible to obtain recombination between loxP sites, specifically in these cells.
The subject of the present invention is also a vector for transferring a DNA fragment into human or animal, in particular mammalian, host cells, characterized in that it comprises a said DNA fragment to be transferred comprising recognition sites specific for the recombinase, in particular two loxP sites, oriented as direct repeats and a cassette for expression of a fusion gene according to the invention. Appropriately, the two sites are placed at each end of said DNA fragment.
Preferably, the fusion gene is placed under the control of expression elements specific for the host cells; in particular, it comprises the sequence of the plasmid pCre-LBD[GR(I747/T)] of SEQ ID NO: 3.
In the excision method according to the invention, said DNA fragment to be excised may be transferred into the host cells before, at the same time, or after the step of introducing the fusion protein or a transfer vector.
The transfer vector according to the invention may be a plasmid or a viral vector. The mode of transfer varies according to the type of target cell and the desired efficiency.
When the material is transferred into the germ cells, microinjection of the male pronucleus of a fertilized oocyte is preferably used. To introduce the DNA into pluripotent embryonic cells (ES cells), the appropriate technique will instead be electroporation or the use of retroviral vectors.
For experiments on somatic cells, it is sought to obtain a maximum efficiency, hence the preferential use of viral vectors: mainly retroviruses and adenoviruses. These viruses should be defective, in particular for application in human health to prevent any multiplication or any risk of revertion.
After integration or otherwise of this type of vectors into the genome, the Cre-lox system makes it possible to excise certain viral sequences which might possibly present a risk in relation to the subsequent propagation of the virus. This is very advantageous for the use of such vectors in gene therapy. Vectors containing a cassette for expression of the Cre-LBD[GR(I747/T)] gene and lox sites delimiting a potentially xe2x80x9cdangerousxe2x80x9d gene and this cassette make it possible, where appropriate after integration into the genome of the recombinant virus, to eliminate these elements. Conversely, it is also possible to activate a gene which has been integrated in an inactive form. This activation may result from the deletion of a fragment from said gene, for example a stop codon or a polyadenylation signal delimited by loxP sites.
When it is desired to transfer genetic material into a cell, constructs are generally used which comprise the gene to be transferred, to which a helper gene has optionally been added, conferring a selective advantage (for example the neo gene conferring resistance to the antibiotic G418).
Selectable markers may also be excised by the excision method according to the invention.
By using conventional techniques, it is possible to modify, by homologous recombination, mammalian, in particular mouse, genes. A loxP site may be introduced into a gene which it is desired to modify, or two loxP sites may delimit sequences which it is desired to modify. In particular, the selectable marker used to make it possible to identify the homologous recombination events may be problematic, and may be removed in a first instance, if necessary, if it is itself delimited by recombinase recognition sites such as loxP or FRT sites, by microinjecting the corresponding recombinase protein, by transfecting the cells, in particular ES cells, with an expression vector for a recombinase protein, or by crossing mice carrying the selectable marker with mice expressing the desired recombinase, in particular the Cre recombinase. This makes it possible to obtain mice whose sole modification in the modified locus is the insertion of recognition sites such as loxP. These mice may subsequently be crossed with mice expressing Cre-LBD[GR(I747/T)] and the modification of the locus obtained by treatment with the ligand such as Dex. This makes it possible to inactivate or modify a gene at a given time, and therefore to study the function of these genes at various times of development. This is particularly advantageous for studying the genes which are essential for the good progress of embryonic development. Indeed, in some cases, conventional homologous recombination techniques cause the death of the embryo and do not allow the function of the gene to be studied at later stages.
The transfer of genes into a given cell forms the basis of gene therapy. The most efficient vectors in this regard are viral vectors, in particular retroviral or adenoviral vectors (39). The subject of the present invention is therefore also a transfer vector according to the invention comprising said DNA fragment to be transferred comprising two loxP sites oriented as a direct repeat, for use as a medicament in gene therapy, when it is administered in combination with a said synthetic glucocorticoid ligand such as dexamethasone.
This type of vector makes it possible to transfer the desired DNA fragment comprising two recombinase specific recognition sites into a gene so that it will be possible for the DNA sequences situated between the two loxP sites to be conditionally excised after administration of said synthetic glucocorticoid
The present invention provides, if addition, human or animal, in particular mammalian, cells transfected with a vector for expression and transfer of a fusion protein according to the invention, or human or animal, in particular mammalian, cells into which a DNA fragment has been transferred with the aid of a transfer vector according to the invention, or alternatively human or animal, in particular mammalian, cells constitutively expressing the fusion protein according to the invention.
A further subject of the present invention is finally a transgenic animal, in particular a mouse, comprising a functional gene for the fusion protein according to the invention, said gene being in particular integrated into one of its chromosomes.
Alternatively, a transgenic animal comprising a functional gene for the fusion protein according to the invention, in which a gene of interest is modified by insertion of loxP site(s), in particular integrated into one or more chromosomes.
The subject of the present invention is finally the use of an expression or transfer vector of cells or of an animal according to the invention as a tool for analyzing or studying the function of a given gene of a host cell and a method of treating cells ex vivo or in vitro involving the use of a method of excision, insertion, inversion or translocation according to the invention and the use of an expression or transfer vector according to the invention.
Other characteristics and advantages of the present invention will emerge in the light of the detailed description which follows.
SEQ ID NO: 1 represents the nucleotide and peptide sequences of GR (I747/T) with the DBD corresponding to aa 421-487 and the LBD corresponding to aa 532-777;
SEQ ID NO: 2 represents the nucleotide and peptide sequences of Cre-LBD[GR(I747/T)].
Nucleotides 1-1029 code for the Cre recombinase (aa 1-343).
Nucleotides 1036-1131 code for the C-terminal region of the D region or the human glucocorticoid receptor [aa 500-531 of GR (I747/T); aa 346-377 of Cre-LBD[GR(I747/T)].
Nucleotides 1132-1869 code for the LBD of GR (I747/T) [aa 532-777 of GR (I747/T); aa 378-623 of Cre-LBD[GR(I747/T)].
Nucleotides 1876-2001 code for the F region of the human estrogen receptor [aa 554-595 of HEGO; aa 626-667 of Cre-LBD[GR(I747/T)].
Nucleotides 1777-1779 correspond to the mutation which replaces isoleucine with a threonine.
SEQ ID NO: 3 represents the nucleotide sequence of the plasmid pCre-LBD[GR(I747/T)].
SEQ ID NOS: 4 to 17 represent the nucleotide sequences of the various oligonucleotides used according to the invention.
FIG. 1 [SEQ ID NOS.: 20-21] is a schematic representation of Cre recombinase activities.
FIG. 2 represents the transcriptional activity of the mutant and wild-type GR receptor in response to dexamethasone and the capacity for attachment of dexamethasone to the two receptors.
FIG. 3 represents a Scatchard analysis of the binding of [3H]-dexamethasone to the wild-type and I747/T mutant receptors.
FIG. 4 represents competition by RU 486 for the Dex binding and the transactivation activities of the wild-type (WT) and GR (I747/T) receptor.
FIG. 5 is a schematic representation of the Cre, hER, Cre-ER, hGR and Cre-LBD[GR(I747/T)] proteins.
FIG. 6 is a schematic representation of the plasmid pCre-LBD[GR(!747/T)].
FIGS. 7A-D represents the nucleotide sequence of pCre-LBD[GF(I747/T)](SEQ ID NO: 5) and the peptide sequence of Cre-LBD[GF(I747/T)] (SEQ ID NO. 5.
FIG. 8 represents the PCR strategy used to detect the wild-type, mutated and recombinant alleles in the test for recombinase activity of Cre-LBD[GR(I747/T)].
FIG. 9 represents the PCR analysis of the excision of the sequences situated between the loxP sites after transient transfection of pCre-LBD[GR(I747/T)] in the murine cells RXRxcex1+/xe2x88x92(LNL) without addition of ligand, or treated with cortisol (10xe2x88x926 M) or with dexamethasone (10xe2x88x926 M).
FIG. 10 represents the PCR analysis of the recombinase activity of Cre-GR(I747/T) in the cells RXRxcex1+/xe2x88x92(LNL):Cre-GR(I747/T) not treated or treated with cortisol or dexamethasone at 10xe2x88x926 M.
FIG. 11 summarizes the recombinase activity of Cre-LBD[GR(I747/T)] in the RXRxcex1+/xe2x88x92(LNL) line constitutively expressing the recombinase, in the presence of increasing concentrations of various ligands.
FIG. 12 represents the PCR analysis of the recombinase activity of Cre-LBD[GR(I747/T)] induced by synthetic ligands in spite of the presence of natural ligands.
FIG. 13 represents the test for the recombinase activity of Cre-LBD[GR(I747/T)] in human HeLa cells.
FIG. 14 is a schematic representation of the trangene Cre-LBD[GR(I747/T)]. Part (a) represents the DNA fragment containing the sequence coding for the Cre-LBD[GR(I747/T) ] gene used to establish transgenic mice and part (b) represents the genomic structure of the wild-type allele RXRxcex1, of the target allele RXRxcex1xcex94AF1 (LNL) and of the excised allele RXRxcex1xcex94AF1 (L) as well as the PCR strategy for analyzing the excision of the marker.
FIG. 15 represents the PCR analysls of the recombinase activity of Cre-LBD[GR(I747/T)] in mice treated with dexamethasone.